Cell-free protein expression offers a rapid, reliable technique for synthesis of proteins. Successfully synthesizing large quantities of high-quality product requires finding the optimal protein expression conditions, such as the type of cellular extract used. Lysate choice for the cell- free reaction can affect expression feasibility, yield and cost. Which extract gives the best results? Here, we compare two of the most common: E. coli and wheat germ.
The cell-free reaction is basically transcription and translation occurring in a test tube outside of the cell using the cellular machinery from inside. Hence, the technique relies on cellular extracts to provide the apparatus required for “cell free” protein expression. This cellular lysate is cleared out of DNA and mRNA, then supplemented with an energy supply and additives such as RNA polymerase to facilitate synthesis of proteins. The origin of the extract can be a variety of sources including prokaryotic, archaeal, fungal, plant, insect, mammalian or bacterial cells.
Different cell-extracts can be used with cell-free expression but at the end E.Coli is the best value for money.
The source of the extract used can have an impact on your product, depending on the type of protein you want to express. The various parameters to consider include post-translational modifications, yield requirement or cost of cell extract preparation. Whilst commercial kits tend to use extracts from Escherichia coli, rabbit reticulocytes or wheat germ, recent studies have compared the different options to provide an evaluation of the choices available. Comparing all possible lysates, they have shown that both E. coli and wheat germ are the front runners in terms of protein yields.
Working with wheat germ:
Wheat germ extract is widely understood as it has been used in cell-free systems since 1973. Over the years, it was optimized by introducing additives to ensure correct disulfide bonding or to include isotopic labels. Thus, the system has proven effective for producing correctly folded or labelled products.
A downfall of wheat germ is in the preparation of the extract. Wheat germ embryos require several costly washing steps during extraction to remove enzymes that inhibit protein expression machinery in the cell-free reaction. This can be counteracted, by the addition of dog pancreas for example, however this further amplifies the already accumulating cost of using the extract.
Expressing with E. coli extract:
As one of the first extracts used, E. coli has been the subject of much development and optimization. Access to a reliable energy source can be a limiting factor in a cell-free system. In 2007, an important advancement in the technology was achieved when fructose-1.6-bisphosphate was discovered. It is as a highly effective, low cost replacement for previously used energy sources making E. coli systems much cheaper to run ever since.
An advantage of E. coli over wheat germ is that the procedure has been scaled up. In wheat germ, large scale-ups have not yet been trialed and therefore industrial quantities of protein cannot be produced.
E. Coli cell extract is scalable, wheat germ is not.
Extracts go head to head:
At Synthelis, we systematically use Escherichia coli-based systems for cell-free protein expression. Whilst both E. coli and wheat germ extracts have their advantages, we consider that the former is more effective overall. From our experience, E. coli is indeed easier to use, more robust and less expensive. Not to mention flexibility of the E. coli system: altering the additives in the reaction mix can provide a huge scope of possibilities.
Even though both wheat germ and E. coli extracts can produce large quantities of protein, E. coli still comes out tops. It can achieve ten times more yield, with the upper ranges reaching 100 mg/ml. Studies also show that the product is more reproducible and easier to upscale. Finally, there are the cost benefits. Wheat germ requires extra preparation steps and costly additives that make it the less economically favorable choice of the two.
For these reasons, at Synthelis, our custom-made cell-free projects focus on using E. coli extract. Our in-house experts can provide you with the support you need to make the right choice and obtain the highest yield of product possible.
Authors & Sources:
Zemella A, Thoring L, Hoffmeister C, Kubick S. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. Chembiochem. 2015;16(17):2420–2431. doi:10.1002/cbic.201500340